In past years, much attention has focused on the gene networks that regulate early developmental processes, but less attention has been paid to how multiple networks and processes are temporally coordinated. Recently the discovery of the transcriptional activator Zelda (Zld), which binds to CAGGTAG and related sequences present in the enhancers of many early-activated genes in Drosophila, hinted at a mechanism for how batteries of genes could be simultaneously activated. Here we use genome-wide binding and expression assays to identify Zld target genes in the early embryo with the goal of unraveling the gene circuitry regulated by Zld. We found that Zld binds to genes involved in early developmental processes such as cellularization, sex determination, neurogenesis, and pattern formation. In the absence of Zld, many target genes failed to be activated, while others, particularly the patterning genes, exhibited delayed transcriptional activation, some of which also showed weak and/or sporadic expression. These effects disrupted the normal sequence of patterning-gene interactions and resulted in highly altered spatial expression patterns, demonstrating the significance of a timing mechanism in early development. In addition, we observed prevalent overlap between Zld-bound regions and genomic “hotspot” regions, which are bound by many developmental transcription factors, especially the patterning factors. This, along with the finding that the most over-represented motif in hotspots, CAGGTA, is the Zld binding site, implicates Zld in promoting hotspot formation. We propose that Zld promotes timely and robust transcriptional activation of early-gene networks so that developmental events are coordinated and cell fates are established properly in the cellular blastoderm embryo.

The Drosophila genome activator Vielfaltig (Vfl), also known as Zelda (Zld), is thought to prime enhancers for activation by patterning transcription factors (TFs). Such priming is accompanied by increased chromatin accessibility, but the mechanisms by which this occurs are poorly understood. Here, we analyze the effect of Zld on genome-wide nucleosome occupancy and binding of the patterning TF Dorsal (Dl). Our results show that early enhancers are characterized by an intrinsically high nucleosome barrier. Zld tackles this nucleosome barrier through local depletion of nucleosomes with the effect being dependent on the number and position of Zld motifs. Without Zld, Dl binding decreases at enhancers and redistributes to open regions devoid of enhancer activity. We propose that Zld primes enhancers by lowering the high nucleosome barrier just enough to assist TFs in accessing their binding motifs and promoting spatially controlled enhancer activation if the right patterning TFs are present. We envision that genome activators in general will utilize this mechanism to activate the zygotic genome in a robust and precise manner.

Summary Zygotic genome activation (ZGA) is a major genome programming event whereby the cells of the embryo begin to adopt specified fates. Experiments in Drosophila and zebrafish revealed that ZGA depends on transcription factors that provide large-scale control of gene expression by direct and specific binding to gene regulatory sequences [1–5]. Zelda (Zld) plays such a role in the Drosophila embryo, where it was shown to control the action of patterning signals [1, 2], however the mechanisms underlying this effect remain largely unclear. A recent model proposed that Zld binding sites act as quantitative regulators of the spatiotemporal expression of genes activated by Dorsal (Dl), the morphogen that patterns the dorsoventral (DV) axis [6]. We test this model experimentally, using enhancers of brinker (brk) and short gastrulation (sog), both of which are directly activated by Dl, but at different concentration thresholds [7–9]. In agreement with the model, we show that there is a clear positive correlation between the number of Zld binding sites and the spatial domain of enhancer activity. Likewise, the timing of expression could be advanced or delayed. We present evidence that Zld facilitates binding of Dl to regulatory DNA, and that this is associated with increased chromatin accessibility. Importantly, the change in chromatin accessibility is strongly correlated with the change in Zld binding, but not Dl. We propose that the ability of genome activators to facilitate read-out of transcriptional input is key to widespread transcriptional induction during ZGA.

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In China, excessive use of nitrogen fertilizers in glasshouses leads to nitrate accumulations in soil and plants, which then limits productivity. Melatonin, an evolutionarily highly conserved molecule, has a wide range of functions in plants. We analyzed the effects of melatonin pretreatment on the growth, mineral nutrition, and nitrogen metabolism in cucumber (Cucumis sativus L. "Jin You No. 1") when seedlings were exposed to nitrate stress. An application of 0.1 mmol/L melatonin significantly improved the growth of plants and reduced their susceptibility to damage due to high nitrate levels (0.6 mol/L) during the ensuing period of stress treatment. Although excess nitrate led to an increase in the concentrations of nitrogen, potassium, and calcium, as well as a decrease in levels of phosphorus and magnesium, exogenous melatonin generally had the opposite effect except for a further rise in calcium concentrations. Pretreatment also significantly reduced the accumulations of nitrate nitrogen and ammonium nitrogen and enhanced the activities of enzymes involved in nitrogen metabolism. Expression of Cs-NR and Cs-GOGAT, two genes that function in that metabolism, was greatly down-regulated when plants were exposed to 0.6 mol/L nitrate, but was up-regulated in plants that had received the 0.1 mmol/L melatonin pretreatment. Our results are the first evidence that melatonin has an important role in modulating the composition of mineral elements and nitrogen metabolism, thereby alleviating the inhibitory effect on growth normally associated with nitrate stress.

Pervasive transcription of the mammalian genome generates numerous long noncoding RNAs (lncRNAs), which are of crucial importance in diverse biological processes. Recent advances in high throughput sequencing technology have helped to accelerate the pace of lncRNA discovery. However, no study on the overall expression patterns of lncRNAs during muscle development has been conducted. We reported here the first analysis of lncRNA landscape in bovine embryonic, neonatal and adult skeletal muscle using Ribo-Zero RNA-Seq, a technology which can capture both poly(A) and poly(A) transcripts. We finally defined 7692 high-confidence lncRNAs and uncovered 401 lncRNAs differentially expressed among three developmental stages, including lncMD, a novel muscle-specific lncRNA which is gradually up-regulated during myoblast differentiation. lncMD overexpression upregulated, whereas lncMD silencing decreased the expression of two well-established myogenic markers, myosin heavy chain (MHC) and myogenin (MyoG). In-depth analyses showed that lncMD acts as a molecular sponge for miR-125b and that insulin-like growth factor 2 (IGF2) is a direct target of miR-125b in cattle. Moreover, lncMD level was positively correlated with IGF2 mRNA level in bovine muscle tissues, a vital corollary to ceRNA function. Altogether, our research showed that lncMD acts as a ceRNA to sequester miR-125b, leading to heightened IGF2 expression and thus promotes muscle differentiation. Our findings also complement the reference genome annotation of cattle, which will likely be useful for further functional lncRNA cloning and more comprehensive studies on lncRNA regulation in muscle development.

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1,2,3-Trisubstituted closo-dodecaborates with B-O, B-N, and B-C bonds as well as a fused borane oxazole ring have been synthesized by rhodium-catalyzed direct cage B-H alkenylation and annulation of ureido boranes in the first reported example of regioselective B-H bond functionalization of the [B H ] cage by transition-metal catalysis. This reaction proceeded at room temperature under ambient conditions and exhibited excellent selectivity for efficient monoalkenylation with good functional-group tolerance. The urea moiety enabled B-H activation by acting as a directing group, was incorporated in the oxazole ring in situ, and also avoided multiple alkenylation. A possible mechanism is proposed on the basis of the isolation of a rhodium agostic intermediate and control experiments.